Three-dimensional culture of pancreatic parenchymal cells cultured living stromal tissue prepared in vitro

ABSTRACT

A stromal cell-based three-dimensional cell culture system is prepared which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time. The stromal cells and connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope a framework composed of a biocompatible non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells. The living stromal tissue so formed provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture and/or cultures implanted in vivo. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts in vivo, which can be utilized in the body as a corrective tissue. For example, and not by way of limitation, the three-dimensional cultures can be used to form tubular tissue structures, like those of the gastrointestinal and genitourinary tracts, as well as blood vessels; tissues for hernia repair and/or tendons and ligaments; etc.

This is a Continuation of U.S. patent application Ser. No. 09/237,980,filed Jan. 25, 1999; which is a continuation of Ser. No. 08/487,749,filed Jun. 7, 1995 (U.S. Pat. No. 5,863,531); which is aContinuation-In-Part of Ser. No. 08/254,096, filed Jun. 6, 1994(abandoned); which is a Continuation-In-Part of Ser. No. 08/131,361,filed Oct. 4, 1993, U.S. Pat. No. 5,443,950; which is a divisional ofSer. No. 07/575,518, filed Aug. 30, 1990, U.S. Pat. No. 5,266,480; whichis a division of Ser. No. 07/402,104, filed Sep. 1, 1989, U.S. Pat. No.5,032,508; which is a continuation-in-part of Ser. No. 07/242,096, filedSep. 8, 1988, U.S. Pat. No. 4,963,489; which is a continuation-in-partof Ser. No. 07/038,110, filed Apr. 14, 1987, (abandoned); which is acontinuation-in-part of Ser. No. 07/036,154, filed Apr. 3, 1987, U.S.Pat. No. 4,721,096; which is a continuation of Ser. No. 06/853,569,filed Apr. 18, 1986, now abandoned, each of which is incorporated byreference herein in its entirety.

TABLE OF CONTENTS

1. INTRODUCTION

2. BACKGROUND OF THE INVENTION

2.1. LONG TERM CELL CULTURE

2.2. CORRECTION OF DEFECTS IN THE BODY

3. SUMMARY OF THE INVENTION

3.1. DEFINITIONS AND ABBREVIATIONS

4. DESCRIPTION OF THE FIGURES

5. DETAILED DESCRIPTION OF THE INVENTION: THE THREE-DIMENSIONAL CELLCULTURE SYSTEM

5.1. ESTABLISHMENT OF THREE-DIMENSIONAL STROMAL TISSUE

5.2. INOCULATION OF TISSUE-SPECIFIC CELLS ONTO THREE-DIMENSIONAL STROMALMATRIX AND MAINTENANCE OF CULTURES

5.3. USES OF THE TRANSPLANTABLE TISSUE GRAFTS GROWN IN THREE-DIMENSIONALCULTURE SYSTEM

5.3.1. TRANSPLANTATION IN VIVO

5.3.2. SCREENING EFFECTIVENESS AND CYTOTOXICITY OF COMPOUNDS IN VITRO

5.3.3. GENE THERAPY

6. TUBULAR BIOLOGICAL TISSUES

6.1. SINGLE MESH LAYER TUBES

6.1.1.. FLAT MESH STARTING MATERIAL

6.1.2. TUBULAR MESH STARTING MATERIAL

6.2. MULTIPLE MESH LAYERS TUBES

6.2.1. MULTIPLE FLAT MESHES

6.2.2. FLAT MESHES WRAPPED AROUND TUBULAR MESHES

6.2.3. MULTIPLE TUBULAR MESHES

7. BLOOD VESSELS

7.1. ARTERIES

7.2. VEINS

8. GASTROINTESTINAL TRACT

9. GENITOURINARY TRACT

9.1. URETER

9.2. URETHRA

10. HERNIA REPAIR

11. FORMATION OF TENDONS AND LIGAMENTS

1. INTRODUCTION

The present invention relates to a stromal cell-based three-dimensionalcell and tissue culture system and its use to form corrective structuresthat can be implanted and utilized in vivo. This culture system can beused for the long term proliferation of cells and tissues in vitro in anenvironment that more closely approximates that found in vivo. Theculture system described herein provides for proliferation andappropriate cell maturation to form structures analogous to tissuecounterparts in vivo. In particular, the invention relates to the use ofthe fibroblast-based three-dimensional cell culture system to constructcomplex structures such as, but not limited to, tubular sections ofgastrointestinal and genitourinary tracts, blood vessels, tissues forhernia repair, tendons and ligaments. The three-dimensional cultures canbe implanted in vivo to correct defects in the body.

2. BACKGROUND OF THE INVENTION

Cell culture systems have been used to study cells, expand cellpopulations for additional study, and in the production of recombinantgene products. However, cell culture systems have not been utilized forthe repair of defects or abnormal tissues in the body.

2.1. Long Term Cell Culture

The majority of vertebrate cell cultures in vitro are grown asmonolayers on an artificial substrate bathed in nutrient medium. Thenature of the substrate on which the monolayers grow may be solid, suchas plastic, or semisolid gels, such as collagen or agar. Disposableplastics have become the preferred substrate used in modern-day tissueor cell culture.

Some attempts have been made to use natural substrates related tobasement membrane components. Basement membranes comprise a mixture ofproteins, glycoproteins and proteoglycans that surround most cells invivo. For example, Reid and Rojkund (1979, In, Methods in Enzymology,Vol. 57, Cell Culture, Jakoby & Pasten, eds., New York, Acad. Press,pp.263-278); Vlodavsky et al., (1980, Cell 19:607-617); Yang et al.,(1979, Proc. Natl. Acad. Sci. U.S.A. 76:3401) have used collagen forculturing hepatocytes, epithelial cells and endothelial tissue. Growthof cells on floating collagen (Michalopoulos and Pitot, 1975, Fed. Proc.34:826) and cellulose nitrate membranes (Savage and Bonney, 1978, Exp.Cell Res. 114:307-315) have been used in attempts to promote terminaldifferentiation. However, prolonged cellular regeneration and theculture of such tissues in such systems has not heretofore beenachieved.

Cultures of mouse embryo fibroblasts have been used to enhance growth ofcells, particularly at low densities. This effect is thought to be duepartly to supplementation of the medium but may also be due toconditioning of the substrate by cell products. In these systems, feederlayers of fibroblasts are grown as confluent monolayers which make thesurface suitable for attachment of other cells. For example, the growthof glioma on confluent feeder layers of normal fetal intestine has beenreported (Lindsay, 1979, Nature 228:80).

While the growth of cells in two dimensions is a convenient method forpreparing, observing and studying cells in culture, allowing a high rateof cell proliferation, it lacks the cell-cell and cell-matrixinteractions characteristic of whole tissue in vivo. In order to studysuch functional and morphological interactions, a few investigators haveexplored the use of three-dimensional substrates such as collagen gel(Douglas et al., 1980, In Vitro 16:306-312; Yang et al., 1979, Proc.Natl. Acad. Sci. 76:3401; Yang et al., 1980, Proc. Natl. Acad. Sci.77:2088-2092; Yang et al., 1981, Cancer Res. 41:1021-1027); cellulosesponge alone (Leighton et al., 1951, J. Natl. Cancer Inst. 12:545-561)or collagen coated (Leighton et al., 1968, Cancer Res. 28:286-296); agelatin sponge, Gelfoam (Sorour et al., 1975, J. Neurosurg. 43:742-749).

In general, these three-dimensional substrates are inoculated with thecells to be cultured. Many of the cell types have been reported topenetrate the matrix and establish a "tissue-like" histology. Forexample, three-dimensional collagen gels have been utilized to culturebreast epithelium (Yang et al., 1981, Cancer Res. 41:1021-1027) andsympathetic neurons (Ebendal, 1976, Exp. Cell Res. 98:159-169).Additionally, various attempts have been made to regenerate tissue-likearchitecture from dispersed monolayer cultures. Kruse and Miedema (1965,J. Cell Biol. 27:273) reported that perfused monolayers could grow tomore than ten cells deep and organoid structures can develop inmultilayered cultures if kept supplied with appropriate medium (see alsoSchneider et al., 1963, Exp. Cell Res. 30:449-459 and Bell et al., 1979,Proc. Natl. Acad. Sci. U.S.A. 76:1274-1279); Green (1978, Science200:1385-1388) has reported that human epidermal keratinocytes may formdematoglyphs (friction ridges) if kept for several weeks withouttransfer; Folkman and Haudenschild (1980, Nature 288:551-556) reportedthe formation of capillary tubules in cultures of vascular endothelialcells cultured in the presence of endothelial growth factor and mediumconditioned by tumor cells; and Sirica et al. (1979, Proc. Natl. Acad.Sci U.S.A. 76:283-287; 1980, Cancer Res. 40:3259-3267) maintainedhepatocytes in primary culture for about 10-13 days on nylon meshescoated with a thin layer of collagen. However, the long term culture andproliferation of cells in such systems has not been achieved.

Indeed, the establishment of long term culture of tissues such as bonemarrow has been attempted. Overall the results were disappointing, inthat although a stromal cell layer containing different cell types israpidly formed, significant hematopoiesis could not be maintained forany real time. (For review see Dexter et al., In Long Term Bone MarrowCulture,1984, Alan R. Liss, Inc., pp. 57-96).

2.2. Correction of Defects in the Body

Surgical approaches to correcting defects in the body, in general,involve the implantation of structures made of biocompatible, inertmaterials, that attempt to replace or substitute for the defectivefunction. Non-biodegradable materials will result in permanentstructures that remain in the body as a foreign object. Implants thatare made of resorbable materials are suggested for use as temporaryreplacements where the object is to allow the healing process to replacethe resorbed material. However, these approaches have met with limitedsuccess for the long-term correction of structures in the body. Forexample, the use of a tubular mesh as a surgical corrective device isdescribed in U.S. Pat. No. 4,347,847 of F. C. Usher issued Sep. 7, 1982.This mesh was used neither to generate a specific tissue culture, nor toreconstruct a tubular structure. Rather it was sutured in place in aflattened configuration to join connective tissues together. In U.S.Pat. No. 4,520,821, issued Jun. 4, 1985, Schmidt et al. disclose the useof a tubular mesh to correct defects in the tubular structures of thegenitourinary tract.

The foreign meshes could not fully replace the damaged tissue, sincesmooth muscle does not grow at the treated site. Bell included a smoothmuscle cell layer in his attempt at constructing blood vessels describedin U.S. Pat. No. 4,546,500, issued Oct. 15, 1985. This construction,however, completely lacked elastin, a necessary component of bloodvessels, and relied on a plastic mesh sleeve to provide the strength andelasticity required of blood vessels in vivo, with disappointingresults. Thus, there has remained a need to construct tubular tissuestructures (or constructs) such that they contain the cellular andextracellular components required to carry out the functions of theirnatural counterparts.

3. SUMMARY OF THE INVENTION

The present invention relates to a stromal cell-based three-dimensionalcell culture system which can be used to culture a variety of differentcells and tissues in vitro for prolonged periods of time. Growth ofstromal cells on the three-dimensional framework results in theformation of a three-dimensional living stromal tissue which can beutilized in the body as a corrective structure. For example, and not byway of limitation, the three-dimensional cultures can be used to formtubular structures, like those of the gastrointestinal and genitourinarytracts, as well as blood vessels; tissues for hernia repair; tendons andligaments; etc.

In accordance with the invention, stromal cells, such as fibroblasts,are inoculated and grown on a three-dimensional framework. The frameworkmay be configured into the shape of the corrective structure desired.Stromal cells may also include other cells found in loose connectivetissue such as smooth muscle cells, endothelial cells,macrophages/monocytes, adipocytes, pericytes, reticular cells found inbone marrow stroma, chondrocytes, etc. During growth in vitro thestromal cells deposit their extracellular matrix proteins onto theframework, thus forming a living stromal tissue; i.e., the stromal cellsand connective tissue proteins naturally secreted by the stromal cellsattach to and substantially envelope the framework composed of abiocompatible non-living material formed into a three-dimensionalstructure having interstitial spaces bridged by the stromal cells. Theliving stromal tissue so formed provides the support, growth factors,and regulatory factors necessary to sustain long-term activeproliferation of cells in culture and deposition of appropriate matrixproteins. When grown in this three-dimensional system, the proliferatingcells mature and segregate properly to form components of adult tissuesanalogous to counterparts found in vivo.

The invention is based, in part, on the discovery that growth of stromalcells in three dimensions will sustain active proliferation of cells inculture for longer periods of time than will monolayer systems. This maybe due, in part, to the increased surface area of the three-dimensionalframework which results in a prolonged period of active proliferation ofstromal cells. These proliferating stromal cells elaborate proteins,growth factors and regulatory factors necessary to support the long termproliferation of both stromal and tissue-specific cells inoculated ontothe stromal matrix. In addition, the three-dimensionality of the matrixallows for a spatial distribution which more closely approximatesconditions in vivo, thus allowing for the formation of microenvironmentsconducive to cellular maturation and migration. The growth of cells inthe presence of this support may be further enhanced by adding proteins,glycoproteins, glycosaminoglycans, a cellular matrix, and othermaterials to support itself or by coating the support with thesematerials. The three-dimensional framework can be shaped to assume theconformation of natural organs and their components.

In another embodiment of the invention, the stromal cells can begenetically engineered to express a gene product beneficial forsuccessful and/or improved transplantation. For example, in the case ofvascular grafts, the stromal cells can be genetically engineered toexpress anticoagulation gene products to reduce the risk ofthromboembolism, atherosclerosis, occlusion, or anti-inflammatory geneproducts to reduce risk of failure. For example, the stromal cells canbe genetically engineered to express tissue plasminogen activator (TPA),streptokinase or urokinase to reduce the risk of clotting.Alternatively, the stromal cells can be engineered to expressanti-inflammatory gene products, e.g., peptides or polypeptidescorresponding to the idiotype of neutralizing antibodies for tumornecrosis factor (TNF), interleukin-2 (IL-2), or other inflammatorycytokines. Preferably, the cells are engineered to express such geneproducts transiently and/or under inducible control during thepost-operative recovery period, or as a chimeric fusion protein anchoredto the stromal cell, e.g., a chimeric molecule composed of anintracellular and/or transmembrane domain of a receptor or receptor-likemolecule, fused to the gene product as the extracellular domain.

In another alternative, the stromal cells can be genetically engineeredto "knock out" expression of factors or surface antigens that promoteclotting or rejection. For example, expression of fibrinogen, vonWillebrands factor or any cell surface molecule that binds to theplatelet α2Bβ-3 receptor can be knocked out in the stromal cells toreduce the risk of clot formation. Likewise, the expression of MHC classII molecules can be knocked out in order to reduce the risk of rejectionof the graft.

In yet another embodiment of the invention, the three-dimensionalculture system of the invention may afford a vehicle for introducinggenes and gene products in vivo to assist or improve the results of thetransplantation and/or for use in gene therapies. For example, genesthat prevent or ameliorate symptoms of vascular disease such as thrombusformation, atherosclerosis, inflammatory reactions, fibrosis andcalcification, may be underexpressed or overexpressed in diseaseconditions. Thus, the level of gene activity in the patient may beincreased or decreased, respectively, by gene replacement therapy byadjusting the level of the active gene product in genetically engineeredstromal cells.

In another alternative, the stromal cells can be genetically engineeredto block gene expression necessary for the transition of smooth musclecells to proliferate, migrate and to lead to development of neointimalhyperplasia, e.g., by antisense oligodeoxynucleotide blockade ofexpression of cell division cycle 2 kinase and proliferating cellnuclear antigen. Mann, M. J., et al., 1995, Proc. Natl. Acad. Sci.U.S.A. 92:4502-4506.

The present invention relates to methods and biological tissue, tubularsections or constructs for the treatment, reconstruction and/orreplacement of defects in the body, including, but not limited to,gastrointestinal and genitourinary tracts, blood vessels such asarteries and veins, tissues for hernia repair, tendons and ligaments.

3.1. Definitions and Abbreviations

The following terms used herein shall have the meanings indicated:

Adherent Layer: cells attached directly to the three-dimensional supportor connected indirectly by attachment to cells that are themselvesattached directly to the support.

Stromal Cells: fibroblasts with or without other cells and/or elementsfound in loose connective tissue, including but not limited to,endothelial cells, pericytes, macrophages, monocytes, plasma cells, mastcells, adipocytes, etc.

Tissue-Specific or Parenchymal Cells: the cells which form the essentialand distinctive tissue of an organ as distinguished from its supportiveframework.

Three-Dimensional Framework: a three-dimensional scaffold composed ofany material and/or shape that (a) allows cells to attach to it (or canbe modified to allow cells to attach to it); and (b) allows cells togrow in more than one layer. This support is inoculated with stromalcells to form the living three-dimensional stromal tissue.

Three-Dimensional Stromal Tissue: a three-dimensional framework whichhas been inoculated with stromal cells that are grown on the support.The extracellular matrix proteins elaborated by the stromal cells aredeposited onto the framework, thus forming a living stromal tissue. Theliving stromal tissue can support the growth of tissue-specific cellslater inoculated to form the three-dimensional cell culture.

Three-Dimensional Cell Culture: a three-dimensional living stromaltissue which has been inoculated with tissue-specific cells andcultured. In general, the tissue specific cells used to inoculate thethree-dimensional stromal matrix should include the "stem" cells (or"reserve" cells) for that tissue; i.e., those cells which generate newcells that will mature into the specialized cells that form theparenchyma of the tissue.

The following abbreviations shall have the meanings indicated:

BFU-E=burst-forming unit-erythroid

CFU-C=colony forming unit-culture

CFU-GEMM=colony forming unit-granuloid, erythroid, monocyte,megakaryocyte

EDTA=ethylene diamine tetraacetic acid

FBS=fetal bovine serum

HBSS=Hank's balanced salt solution

HS=horse serum

LTBMC=long term bone marrow culture

MEM=minimal essential medium

PBL=peripheral blood leukocytes

PBS=phosphate buffered saline

RPMI 1640=Roswell Park Memorial Institute medium number 1640 (GIBCO,Inc., Grand Island, N.Y.)

SEM=scanning electron microscopy

4. DESCRIPTION OF THE FIGURES

FIG. 1 is a scanning electron micrograph depicting attachment to thethree-dimensional matrix and extension of cellular processes across themesh opening. Fibroblasts are actively secreting matrix proteins and areat the appropriate stage of subconfluency which should be obtained priorto inoculant with tissue-specific cells.

5. DETAILED DESCRIPTION OF THE INVENTION: THE THREE-DIMENSIONAL CELLCULTURE SYSTEM

The present invention relates to three-dimensional living stromaltissues that can be used as corrective structures in the body,including, but not limited to, tubular structures that can be used toreplace or repair blood vessels, gastrointestinal tract, or urinarytract; filamentous or tubular structures that can be used to replace orrepair tendons and ligaments; and tubular or flat structures that can beused to repair defects such as hernias. The living stromal tissue of theinvention comprises stromal cells grown on a three-dimensionalframework, matrix or network. The three-dimensional framework can beformed into any desired shape; e.g., mesh type frameworks can be used toform tubular structures; rope-like structures can be woven or tubes orfilaments can be used as the framework for growing tendons andligaments, etc.

In previously known tissue culture systems, the cells were grown in amonolayer. Cells grown on a three-dimensional stromal support, inaccordance with the present invention, grow in multiple layers, forminga cellular matrix. This matrix system approaches physiologic conditionsfound in vivo to a greater degree than previously described monolayertissue culture systems. The three-dimensional cell culture system isapplicable to the proliferation of different types of cells andformation of a number of different tissues, including but not limited tobone marrow, skin, liver, pancreas, kidney, adrenal and neurologictissue, as well as tissues of the gastrointestinal and genitourinarytracts, and the circulatory system, to name but a few. See U.S. Pat.Nos. 4,721,096; 4,963,489; 5,032,508; 5,266,480; and 5,160,490, each ofwhich is incorporated by reference herein in its entirety.

The stromal cells used in the three-dimensional cultures comprisefibroblasts with or without additional cells and/or elements describedmore fully herein. The fibroblasts and other cells and/or elements thatcomprise the stroma may be fetal or adult in origin, and may be derivedfrom convenient sources such as skin, liver, pancreas, arteries, veins,umbilical cord, and placental tissues, etc. Such tissues and/or organscan be obtained by appropriate biopsy or upon autopsy. In fact, cadaverorgans may be used to provide a generous supply of stromal cells andelements.

Fetal fibroblasts will support the growth of many different cells andtissues in the three-dimensional culture system, and, therefore, can beinoculated onto the matrix to form a "generic" stromal support matrixfor culturing any of a variety of cells and tissues. However, in certaininstances, it may be preferable to use a "specific" rather than"generic" stromal support matrix, in which case stromal cells andelements can be obtained from a particular tissue, organ, or individual.For example, where the three-dimensional culture is to be used forpurposes of transplantation or implantation in vivo, it may bepreferable to obtain the stromal cells and elements from the individualwho is to receive the transplant or implant. This approach might beespecially advantageous where immunological rejection of the transplantand/or graft versus host disease is likely. Moreover, fibroblasts andother stromal cells and/or elements may be derived from the same type oftissue to be cultured in the three-dimensional system. This might beadvantageous when culturing tissues in which specialized stromal cellsmay play particular structural/functional roles; e.g., smooth musclecells of arteries, glial cells of neurological tissue, Kupffer cells ofliver, etc.

Once inoculated onto the three-dimensional support, the stromal cellswill proliferate on the framework and deposit the connective tissueproteins naturally secreted by the stromal cells. The stromal cells andtheir naturally secreted connective tissue proteins substantiallyenvelop the framework thus forming the living stromal tissue which willsupport the growth of tissue-specific cells inoculated into thethree-dimensional culture system of the invention. In fact, wheninoculated with the tissue-specific cells, the three-dimensional stromaltissue will sustain active proliferation of the culture for long periodsof time. Importantly, because openings in the mesh permit the exit ofstromal cells in culture, confluent stromal cultures do not exhibitcontact inhibition, and the stromal cells continue to grow, divide, andremain functionally active.

Growth and regulatory factors may be added to the culture, but are notnecessary since they are elaborated by the stromal tissue. The use ofgrowth factors (for example, but not limited to, αFGF, βFGF, insulingrowth factor or TGF-betas), or natural or modified blood products orother bioactive biological molecules (for example, but not limited to,hyaluronic acid or hormones), even though not absolutely necessary inthe present invention, may be used to further enhance the colonizationof the three-dimensional framework or scaffolding.

Because, according to the invention, it is important to recreate, inculture, the cellular microenvironment found in vivo for a particulartissue, the extent to which the stromal cells are grown prior to use ofthe cultures in vivo may vary depending on the type of tissue to begrown in three-dimensional tissue culture. The living stromal tissuesmay be used as corrective structures by implanting them in vivo.Alternatively, the living stromal tissues may be inoculated with anothercell type and implanted in vivo, with or without prior culturing invitro. In addition, the stromal cells grown in the system may begenetically engineered to produce gene products beneficial totransplantation, e.g., anti-inflammatory factors, e.g., anti-GM-CSF,anti-TNF, anti-IL-1, anti-IL-2, etc. Alternatively, the stromal cellsmay be genetically engineered to "knock out" expression of native geneproducts that promote inflammation, e.g., GM-CSF, TNF, IL-1, IL-2, or"knock out" expression of MHC in order to lower the risk of rejection.In addition, the stromal cells may be genetically engineered for use ingene therapy to adjust the level of gene activity in a patient to assistor improve the results of the tubular tissue transplantation.

In another alternative, the stromal cells can be genetically engineeredto block gene expression necessary for the transition of smooth musclecells to proliferate, migrate and to lead to development of neointimalhyperplasia, e.g., by antisense oligodeoxynucleotide blockade ofexpression of cell division cycle 2 kinase and proliferating cellnuclear antigen.

The invention is based, in part, upon the discovery that growth of thestromal cells in three dimensions will sustain active proliferation ofboth the stromal and tissue-specific cells in culture for much longertime periods than will monolayer systems. Moreover, thethree-dimensional system supports the maturation, differentiation, andsegregation of cells in culture in vitro to form components of adulttissues analogous to counterparts found in vivo.

In yet another application, the three-dimensional tubular tissue orconstruct may be grown within a "bioreactor" to produce grafts populatedwith viable human cells. For example, but not limited to, a vasculargraft, which may be assembled as a three-dimensional framework andhoused in the treatment chamber of the bioreactor. Applying radialstress to the vascular graft located in the treatment chamber duringseeding and culturing results in a vascular graft with cells and theirfibers oriented so as to more likely tolerate the physiologicalconditions found in the human body. In this manner, the "bioreactor"creates a dynamic environment in which to seed and culturetissue-engineered vascular or other biological grafts or otherimplantable constructs.

Although the applicants are under no duty or obligation to explain themechanism by which the invention works, a number of factors inherent inthe three-dimensional culture system may contribute to its success:

(a) The three-dimensional framework provides a greater surface area forprotein attachment, and consequently, for the adherence of stromalcells; and

(b) Because of the three-dimensionality of the framework, stromal cellscontinue to grow actively, in contrast to cells in monolayer cultures,which grow to confluence, exhibit contact inhibition,.and cease to growand divide. The elaboration of growth and regulatory factors byreplicating stromal cells may be partially responsible for stimulatingproliferation and regulating differentiation of cells in culture;

(c) The three-dimensional framework allows for a spatial distribution ofcellular elements which is more analogous to that found in thecounterpart tissue in vivo;

(d) The increase in potential volume for cell growth in thethree-dimensional system may allow the establishment of localizedmicroenvironments conducive to cellular maturation;

(e) The three-dimensional framework maximizes cell-cell interactions byallowing greater potential for movement of migratory cells, such asmacrophages, monocytes and possibly lymphocytes in the adherent layer;

(f) It has been recognized that maintenance of a differentiated cellularphenotype requires not only growth/differentiation factors but also theappropriate cellular interactions. The present invention effectivelyrecreates the tissue microenvironment.

The three-dimensional stromal tissues, the culture system itself, andits maintenance, as well as various uses of the three-dimensionalcultures are described in greater detail in the subsections below.

5.1. Establishment of Three-Dimensional Stromal Tissue

The three-dimensional support or framework may be of any material and/orshape that: (a) allows cells to attach to it-(or can be modified toallow cells to attach to it); and (b) allows cells to grow in more thanone layer. A number of different materials may be used to form theframework, including but not limited to: non-biodegradable materials,e.g., nylon (polyamides), dacron (polyesters), polystyrene,polypropylene, polyacrylates, polyvinyl compounds (e.g.,polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE;teflon), thermanox (TPX), nitrocellulose, cotton; and biodegradablematerials, e.g., polyglycolic acid (PGA), collagen, collagen sponges,cat gut sutures, cellulose, gelatin, dextran, polyalkanoates, etc. Anyof these materials may be woven braided, knitted, etc., into a mesh, forexample, to form the three-dimensional framework. The framework, in turncan be fashioned into any shape desired as the corrective structure,e.g., tubes, ropes, filaments, etc. Certain materials, such as nylon,polystyrene, etc., are poor substrates for cellular attachment. Whenthese materials are used as the three-dimensional framework, it isadvisable to pre-treat the framework prior to inoculation of stromalcells in order to enhance the attachment of stromal cells to thesupport. For example, prior to inoculation with stromal cells, nylonframeworks could be treated with 0.1M acetic acid, and incubated inpolylysine, FBS, and/or collagen to coat the nylon. Polystyrene could besimilarly treated using sulfuric acid.

For implantation of the three-dimensional culture in vivo, it may bepreferable to use biodegradable matrices such as polyglycolic acid,collagen, collagen sponges, woven collagen, catgut suture material,gelatin, polylactic acid, or polyglycolic acid and copolymers thereof,for example. Where the cultures are to be maintained for long periods oftime or cryopreserved, non-degradable materials such as nylon, dacron,polystyrene, polyacrylates, polyvinyls, teflons, cotton, etc., may bepreferred. A convenient nylon mesh which could be used in accordancewith the invention is Nitex, a nylon filtration mesh having an averagepore size of 210 μm and an average nylon fiber diameter of 90 μm(#3-210/36, Tetko, Inc., New York).

Stromal cells comprising fibroblasts, with or without other cells andelements described below, are inoculated onto the framework. Thesefibroblasts may be derived from organs, such as skin, liver, pancreas,etc., which can be obtained by biopsy (where appropriate) or uponautopsy. In fact fibroblasts can be obtained in quantity ratherconveniently from any appropriate cadaver organ. As previouslyexplained, fetal fibroblasts can be used to form a "generic"three-dimensional stromal matrix that will support the growth of avariety of different cells and/or tissues. However, a "specific" stromaltissue may be prepared by inoculating the three-dimensional frameworkwith fibroblasts derived from the same type of tissue to be culturedand/or from a particular individual who is later to receive the cellsand/or tissues grown in culture in accordance with the three-dimensionalsystem of the invention.

Fibroblasts may be readily isolated by disaggregating an appropriateorgan or tissue which is to serve as the source of the fibroblasts. Thismay be readily accomplished using techniques known to those skilled inthe art. For example, the tissue or organ can be disaggregatedmechanically and/or treated with digestive enzymes and/or chelatingagents that weaken the connections between neighboring cells making itpossible to disperse the tissue into a suspension of individual cellswithout appreciable cell breakage. Enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withany of a number of digestive enzymes either alone or in combination.These include but are not limited to trypsin, chymotrypsin, collagenase,elastase, and/or hyaluronidase, DNase, pronase, dispase etc. Mechanicaldisruption can also be accomplished by a number of methods including,but not limited to, the use of grinders, blenders, sieves, homogenizers,pressure cells, or insonators to name but a few. For a review of tissuedisaggregation techniques, see Freshney, Culture of Animal Cells. AManual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch.9, pp. 107-126.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thefibroblasts and/or other stromal cells and/or elements can be obtained.This also may be accomplished using standard techniques for cellseparation including, but not limited to, cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counterstreaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting. For a review of clonal selection and cell separationtechniques, see Freshney, Culture of Animal Cells. A Manual of BasicTechniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137-168.

The isolation of fibroblasts may, for example, be carried out asfollows: fresh tissue samples are thoroughly washed and minced an Hanksbalanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated fibroblasts can then be grown to confluency, liftedfrom the confluent culture and inoculated onto the three-dimensionalmatrix (see, Naughton et al., 1987, J. Med. 18 (3 and 4) 219-250).Inoculation of the three-dimensional framework with a high concentrationof stromal cells, e.g., approximately 10 sup 6 to 5×10 sup 7 cells/ml,will result in the establishment of the three-dimensional stromal tissuein shorter periods of time.

In addition to fibroblasts, other cells may be added to form thethree-dimensional stromal tissue. For example, other cells found inloose connective tissue may be inoculated onto the three-dimensionalsupport along with fibroblasts. Such cells include but are not limitedto smooth muscle cells, endothelial cells, pericytes, macrophages,monocytes, plasma cells, mast cells, adipocytes, etc. These stromalcells may readily be derived from appropriate organs such as arteries,skin, liver, etc., using methods known in the art such as thosediscussed above. In one embodiment of the invention, stromal cells whichare specialized for the particular tissue to be cultured may be added tothe fibroblast stroma. For example, stromal cells of hematopoietictissue, including but not limited to fibroblasts, endothelial cells,macrophages/monocytes, adipocytes and reticular cells, could be used toform the three-dimensional subconfluent stroma for the long term cultureof bone marrow in vitro. Hematopoietic stromal cells may be readilyobtained from the "buffy coat" formed in bone marrow suspensions bycentrifugation at low forces, e.g., 3000×g. In the stromal layer thatmakes up the inner wall of arteries, a high proportion ofundifferentiated smooth muscle cells can be added to provide the proteinelastin. Stromal cells of liver may include fibroblasts, Kupffer cells,and vascular and bile duct endothelial cells. Similarly, glial cellscould be used as the stroma to support the proliferation of neurologicalcells and tissues; glial cells for this purpose can be obtained bytrypsinization or collagenase digestion of embryonic or adult brian(Ponten and Westermark, 1980, in Federof, S. Hertz, L., eds, "Advancesin Cellular Neurobiology," Vol. 1, New York, Academic Press, pp.209-227). Again, where the cultured cells are to be used fortransplantation or implantation in vivo it is preferable to obtain thestromal cells from the patient's own tissues. The growth of cells in thethree-dimensional stromal cell culture may be further enhanced by addingto the framework, or coating the support with proteins (e.g., collagens,elastic fibers, reticular fibers) glycoproteins, glycosaminoglycans(e.g., heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate,dermatan sulfate, keratin sulfate, etc.), a cellular matrix, and/orother materials.

The stromal cells may be inoculated onto the framework before or afterforming the shape desired for implantation, e.g., tubes, ropes,filaments. After inoculation of the stromal cells, the three-dimensionalframework should be incubated in an appropriate nutrient medium. Manycommercially available media such as RPMI 1640, Fisher's, Iscove's,McCoy's, and the like may be suitable for use. It is important that thethree-dimensional stromal cell cultures be suspended or floated in themedium during the incubation period in order to maximize proliferativeactivity. In addition, the culture should be "fed" periodically toremove the spent media, depopulate released cells, and add fresh media.

During the incubation period, the stromal cells will grow linearly alongand envelop the three-dimensional framework before beginning to growinto the openings of the framework. It is important to grow the cells toan appropriate degree which reflects the amount of stromal cells presentin the in vivo tissue prior to inoculation of the stromal matrix withthe tissue-specific cells.

The openings of the framework should be of an appropriate size to allowthe stromal cells to stretch across the openings. Maintaining activelygrowing stromal cells which stretch across the framework enhances theproduction of growth factors which are elaborated by the stromal cells,and hence will support long term cultures. For example, if the openingsare too small, the stromal cells may rapidly achieve confluence but beunable to easily exit from the mesh; trapped cells may exhibit contactinhibition and cease production of the appropriate factors necessary tosupport proliferation and maintain long term cultures. If the openingsare too large, the stromal cells may be unable to stretch across theopening; this will also decrease stromal cell production of theappropriate factors necessary to support proliferation and maintain longterm cultures. When using a mesh type of support, as exemplified herein,we have found that openings ranging from about 150 μm to about 220 μmwill work satisfactorily. However, depending upon the three-dimensionalstructure and intricacy of the framework, other sizes may work equallywell. In fact, any shape or structure that allow the stromal cells tostretch and continue to replicate and grow for lengthy time periods willwork in accordance with the invention.

Different proportions of the various types of collagen deposited on thesupport can also affect the growth of tissue-specific or other cellswhich may be later inoculated onto the stromal tissue or which may growonto the structure in vivo. For example, for optimal growth ofhematopoietic cells, the matrix should preferably contain collagen typesIII, IV and I in an approximate ratio of 6:3:1 in the initial matrix.For three-dimensional skin culture systems, collagen types I and III arepreferably deposited in the initial matrix. The proportions of collagentypes deposited can be manipulated or enhanced by selecting stromalcells which elaborate the appropriate collagen type and inoculating suchstromal cells onto the framework. For example, fibroblasts can beselected using monoclonal antibodies of an appropriate isotype orsubclass that is capable of activating complement, and which defineparticular collagen types. These antibodies and complement can be usedto select negatively the fibroblasts which express the desired collagentype. Alternatively, the stroma used to inoculate the matrix can be amixture of cells which synthesize the appropriate collagen typesdesired. The distribution and origins of the five types of collagen isshown in Table I.

                  TABLE I                                                         ______________________________________                                        DISTRIBUTIONS AND ORIGINS OF THE FIVE                                          TYPES OF COLLAGEN                                                              Collagen   Principal        Cells of                                                                       Type    Tissue Distribution                    ______________________________________                                                                      Origin                                          I        Loose and dense ordinary                                                                       Fibroblasts                                                 connective tissue;         and reticular                                      collagen fibers            cells; smooth                                      Fibrocartilage             muscle cells                                       Bone                       Osteoblast                                         Dentin                     Odontoblasts                                 II    Hyaline and elastic         Chondrocytes                                       cartilage                                                                     Vitreous body of eye       Retinal cells                               III    Loose connective tissue;    Fibroblasts and                                   reticular fibers           reticular cells                                    Papillary layer of dermis                                                     Blood vessels              Smooth muscle cells;                                                          endothelial cells                           IV    Basement membranes          Epithelial and                                                                endothelial cells                                  Lens capsule of eye        Lens fibers                                 V    Fetal membranes; placenta    Fibroblasts                                        Basement membranes                                                            Bone                                                                          Smooth muscle              Smooth muscle  cells                      ______________________________________                                    

Thus, depending upon the tissue to be cultured and the collagen typesdesired, the appropriate stromal cell(s) may be selected to inoculatethe three-dimensional matrix.

Similarly, the relative amounts of collagenic and elastic fibers presentin the stromal layer can be modulated by controlling the ratio ofcollagen producing cells to elastin producing cells in the initialinoculum. For example, since the inner walls of arteries are rich inelastin, an arterial stroma should contain a high concentration of theundifferentiated smooth muscle cells which elaborate elastin.

During incubation of the three-dimensional stromal cell cultures,proliferating cells may be released from the matrix. These releasedcells may stick to the walls of the culture vessel where they maycontinue to proliferate and form a confluent monolayer. This should beprevented or minimized, for example, by removal of the released cellsduring feeding, or by transferring the three-dimensional stromal cultureto a new culture vessel. The presence of a confluent monolayer in thevessel will "shut down" the growth of cells in the three-dimensionalmatrix and/or culture. Removal of the confluent monolayer or transfer ofthe culture to fresh media in a new vessel will restore proliferativeactivity of the three-dimensional culture system. Such removal ortransfers should be done in any culture vessel which has a stromalmonolayer exceeding 25% confluency. Alternatively, the culture systemcould be agitated to prevent the released cells from sticking, orinstead of periodically feeding the cultures, the culture system couldbe set up so that fresh media continuously flows through the system. Theflow rate could be adjusted to both maximize proliferation within thethree-dimensional culture, and to wash out and remove cells releasedfrom the culture, so that they will not stick to the walls of the vesseland grow to confluence. In any case, the released stromal cells can becollected and cryopreserved for future use.

The living stromal tissue so formed can be used as a correctivestructure in vivo. Alternatively, other cells, such as parenchymalcells, may be inoculated and grown on the three-dimensional livingstromal tissue prior to implantation in vivo.

5.2. Inocullation of Tissue-Specific Cells onto Three-DimensionalStromal Matrix and Maintenance of Cultures

Once the three-dimensional stromal cell culture has reached theappropriate degree of growth, additional cells such as tissue-specificcells (parenchymal cells) or surface layer cells which are desired to becultured may also be inoculated onto the living stromal tissue. Suchcells inoculated onto the living stromal tissue can be incubated toallow the cells to adhere to the stromal tissue, and implanted in vivowhere continued growth can occur. Alternatively, the cells can be grownon the living stromal tissue in vitro to form a cultured counterpart ofthe native tissue prior to implantation in vivo. A high concentration ofcells in the inoculum will advantageously result in increasedproliferation in culture much sooner than will low concentrations. Thecells chosen for inoculation will depend upon the tissue to be cultured,which may include, but is not limited to, bone marrow, skin, liver,pancreas, kidney, neurological tissue, adrenal gland, mucosalepithelium, endothelium, and smooth muscle, to name but a few.

For example, and not by way of limitation, a variety of epithelial cellscan be cultured on the three-dimensional living stromal tissue. Examplesof such epithelial cells include, but are not limited to, oral mucosaand gastrointestinal (G.I.) tract cells. Such epithelial cells may beisolated by enzymatic treatment of the tissue according to methods knownin the art, followed by expansion of these cells in culture andapplication of epithelial cells to the three-dimensional stromal supportcell matrix (neo-submucosa). The presence of the submucosa providesgrowth factors and other proteins which promote normal division anddifferentiation of themoral mucosa cells and the cells of the G.I. tractlining. Using this methodology, other epithelial cells can be grownsuccessfully, including nasal epithelium, respiratory tract epithelium,vaginal epithelium, and corneal epithelium.

In general, this inoculum should include the "stem" cell (also calledthe "reserve" cell) for that tissue; i.e., those cells which generatenew cells that will mature into the specialized cells that form thevarious components of the tissue.

The parenchymal or other surface layer cells used in the inoculum may beobtained from cell suspensions prepared by disaggregating the desiredtissue using standard techniques described for obtaining stromal cellsin Section 5.1 above. The entire cellular suspension itself could beused to inoculate the three-dimensional living stromal tissue. As aresult, the regenerative cells contained within the homogenate willproliferate, mature, and differentiate properly on the matrix, whereasnon-regenerative cells will not. Alternatively, particular cell typesmay be isolated from appropriate fractions of the cellular suspensionusing standard techniques described for fractionating stromal cells inSection 5.1 above. Where the "stem" cells or "reserve" cells can bereadily isolated, these may be used to preferentially inoculate thethree-dimensional stromal support. For example, when culturing bonemarrow, the three-dimensional stroma may be inoculated with bone marrowcells, either fresh or derived from a cryopreserved sample. Whenculturing skin, the three-dimensional stroma may be inoculated withmelanocytes and keratinocytes. When culturing liver, thethree-dimensional stroma may be inoculated with hepatocytes. Whenculturing pancreas, the three-dimensional stroma may be inoculated withpancreatic endocrine cells. For a review of methods which may beutilized to obtain parenchymal cells from various tissues, see,Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed.,A. R. Liss, Inc., New York, 1987, Ch. 20, pp. 257-288.

During incubation, the three-dimensional cell culture system should besuspended or floated in the nutrient medium. Cultures should be fed withfresh media periodically. Again, care should be taken to prevent cellsreleased from the culture from sticking to the walls of the vessel wherethey could proliferate and form a confluent monolayer. The release ofcells from the three-dimensional culture appears to occur more readilywhen culturing diffuse tissues as opposed to structured tissues. Forexample, the three-dimensional skin culture of the invention ishistologically and morphologically normal; the distinct dermal andepidermal layers do not release cells into the surrounding media. Bycontrast, the three-dimensional bone marrow cultures of the inventionrelease mature non-adherent cells into the medium much the way suchcells are released in marrow in vivo. As previously explained, shouldthe released cells stick to the culture vessel and form a confluentmonolayer, the proliferation of the three-dimensional culture will be"shut down". This can be avoided by removal of released cells duringfeeding, transfer of the three-dimensional culture to a new vessel, byagitation of the culture to prevent sticking of released cells to thevessel wall, or by the continuous flow of fresh media at a ratesufficient to replenish nutrients in the culture and remove releasedcells. In any case, the mature released cells could be collected andcryopreserved for future use.

Growth factors and regulatory factors need not be added to the mediasince these types of factors ate elaborated by the three-dimensionalstromal cells. However, the addition of such factors, or the inoculationof other specialized cells may be used to enhance, alter or modulateproliferation and cell maturation in the cultures. The growth andactivity of cells in culture can be affected by a variety of growthfactors such as insulin, growth hormone, somatomedins, colonystimulating factors, erythropoietin, epidermal growth factor, hepaticerythropoietic factor (hepatopoietin), and liver-cell growth factor.Other factors which regulate proliferation and/or differentiationinclude prostaglandins, interleukins, and naturally-occurring chalones.

5.3. Uses of the Transplantable Tissue Grafts Grown in Three-DimensionalCulture System

The three-dimensional culture system of the invention can be used in avariety of applications. These include but are not limited totransplantation or implantation of either the cultured cells obtainedfrom the matrix, or the cultured matrix itself in vivo. Thethree-dimensional tissue culture implants may, according to theinvention, be used to replace or augment existing tissue, to introducenew or altered tissue, to modify artificial prostheses, or to jointogether biological tissues or structures. For example, and not by wayof limitation, specific embodiments of the invention include but are notlimited to: (i) dental prostheses joined to a three-dimensional cultureof oral mucosa; (ii) tubular three-dimensional tissue implants (such asgastrointestinal tract, genitourinary tract and blood vessels; (iii)ligament or tendon implants; (iv) tissues for hernia repair; and (v)genetically altered cells grown in the three-dimensional culture whichexpress a recombinant gene product.

5.3.1. Transplantation in Vivo

The three-dimensional cultures can be implanted in vivo to correctdefects; replace surgically removed tissues; repair joints; implantshunts; repair hernias; etc. To this end, the living stromal tissueitself could be implanted in vivo. Depending upon the application, theimplant may first be treated to kill the cells in the culture prior toimplantation. For example, when treating conditions where growth factorsmay aggravate a pre-existing condition, e.g., in rheumatoid arthritis,it may be preferred to kill the cells which produce growth factors inthe culture. This can be accomplished after the stromal tissue is formedin vitro but prior to implantation in vivo, by irradiation, or byfreeze-thawing the cultures and washing away components of lysed cells.Alternatively, where enhancement of wound healing is desired, thecultures can be implanted in a viable state so that growth factors areproduced at the implant site. In yet another alternative, other cells,such as parenchymal cells, may be inoculated onto the living stromaltissue prior to implantation in vivo. These cultures may be furthergrown in vitro prior to implantation in vivo.

The basic manifestation of a hernia is a protrusion of the abdominalcontents into a defect within the fascia. Surgical approaches towardhernia repair is focused on reducing the hernial contents into theperitoneal cavity and producing a firm closure of the fascial defecteither by using prosthetic, allogeneic or autogenous materials. A numberof techniques have been used to produce this closure including themovement of autologous tissues and the use of synthetic mesh products.Drawbacks to these current products and procedures include herniarecurrence, where the closure weakens again, allowing the abdominalcontents back into the defect.

Insertion of the cultured invention in hernia repair would be likely viaan open procedure despite trends toward minimally invasive surgeries asthe conversion of herniorrhaphy from open to endoscopic procedures hasproved slow.

In yet another example, ligaments and tendons are viscoelasticstructures that increase in brittleness with age, leading to ligamentoustears. These structures are complex, relatively static collagenousstructures with functional links to the bone, muscle, menisci and othernearby tendons and ligaments. Surgical repair of these structures areconducted via either open procedures or arthroscopically-assistedprocedures. Autografts are typically used from other sites in the knee.However, autografts can cause donor site morbidity. Other materialswhich are used in place of autografts, such as allografts, bovinetendons, polyesters and carbon fiber reinforced polymers, are subject tomechanical failure and can cause immunogenic complications.

5.3.2. Screening Effectiveness and Cytotoxicity of Compounds in Vitro

The three-dimensional cultures may be used in vitro to screen a widevariety of compounds, for effectiveness and cytotoxicity ofpharmaceutical agents, growth/regulatory factors, natural and modifiedblood products, anticoagulants, clotting agents or anti-calcificationagents, etc. To this end, the cultures are maintained in vitro andexposed to the compound to be tested. The activity of a cytotoxiccompound can be measured by its ability to damage or kill cells inculture. This may readily be assessed by vital staining techniques. Theeffect of growth/regulatory factors may be assessed by analyzing thecellular content of the matrix, e.g., by total cell counts, anddifferential cell counts. This may be accomplished using standardcytological and/or histological techniques including the use ofimmunocytochemical techniques employing antibodies that definetype-specific cellular antigens. The effect of various drugs on normalcells cultured in the three-dimensional system may be assessed.

5.3.3. Gene Therapy

The three-dimensional culture system of the invention may afford avehicle for introducing genes and gene products in vivo to assist orimprove the results of the transplantation and/or for use in genetherapies. For example, for vascular grafts, the stromal cells can begenetically engineered to express anticoagulation gene products toreduce the risk of thromboembolism, or anti-inflammatory gene productsto reduce the risk of failure due to inflammatory reactions. In thisregard, the stromal cells can be genetically engineered to express TPA,streptokinase or urokinase to reduce the risk of clotting.Alternatively, for vascular or other types of tissue grafts, the stromalcells can be engineered to express anti-inflammatory gene products, forexample, peptides or polypeptides corresponding to the idiotype ofneutralizing antibodies for TNF, IL-2, or other inflammatory cytokines.Preferably, the cells are engineered to express such gene productstransiently and/or under inducible control during the post-operativerecovery period, or as a chimeric fusion protein anchored to the stromalcells, for example, a chimeric molecule composed of an intracellularand/or transmembrane domain of a receptor or receptor-like molecule,fused to the gene product as the extracellular domain. In anotherembodiment, the stromal cells could be genetically engineered to expressa gene for which a patient is deficient, or which would exert atherapeutic effect, e.g., HDL, apolipoprotein E, etc. The genes ofinterest engineered into the stromal cells need to be related to thedisease being treated. For example, for vascular disease the stromalcells can be engineered to express gene products that are carried by theblood; e.g., cerebredase, adenosine deaminase, α-1-antitrypsin. In aparticular embodiment, a genetically engineered vascular graft cultureimplanted to replace a section of a vein or artery can be used todeliver gene products such as α-1-antitrypsin to the lungs; in such anapproach, constitutive expression of the gene product is preferred.

The stromal cells can be engineered using a recombinant DNA constructcontaining the gene used to transform or transfect a host cell which iscloned and then clonally expanded in the three-dimensional culturesystem. The three-dimensional culture which expresses the active geneproduct, could be implanted into an individual who is deficient for thatproduct. For example, genes that prevent or ameliorate symptoms ofvarious types of vascular, genitourinary tract, hernia orgastrointestinal diseases may be underexpressed or down regulated underdisease conditions. Specifically, expression of genes involved inpreventing the following pathological conditions may be down-regulated,for example: thrombus formation, inflammatory reactions, and fibrosisand calcification of the valves. Alternatively, the activity of geneproducts may be diminished, leading to the manifestations of some or allof the above pathological conditions and eventual development ofsymptoms of valvular disease. Thus, the level of gene activity may beincreased by either increasing the level of gene product present or byincreasing the level of the active gene product which is present in thethree-dimensional culture system. The three-dimensional culture whichexpresses the active target gene product can then be implanted into thevalvular disease patient who is deficient for that product. "Targetgene," as used herein, refers to a gene involved in diseases such as,but not limited to, vascular, genitourinary tract, hernia orgastrointestinal disease in a manner by which modulation of the level oftarget gene expression or of target gene product activity may act toameliorate symptoms of valvular disease.

Further, patients may be treated by gene replacement therapy during thepost-recovery period after transplantation. Tissue constructs or sheetsmay be designed specifically to meet the requirements of an individualpatient, for example, the stromal cells may be genetically engineered toregulate one or more genes; or the regulation of gene expression may betransient or long-term; or the gene activity may be non-inducible orinducible. For example, one or more copies of a normal target gene, or aportion of the gene that directs the production of a normal target geneprotein product with target gene function, may be inserted into humancells that populate the three-dimensional constructs using eithernon-inducible vectors including, but are not limited to, adenovirus,adeno-associated virus, and retrovirus vectors, or inducible promoters,including metallothionein, or heat shock protein, in addition to otherparticles that introduce DNA into cells, such as liposomes or direct DNAinjection or in gold particles. For example, the gene encoding the humancomplement regulatory protein, which prevents rejection of the graft bythe host, may be inserted into human fibroblasts. Nature 375:89 (May,1995).

The three-dimensional cultures containing such genetically engineeredstromal cells, e.g., either mixtures of stromal cells each expressing adifferent desired gene product, or a stromal cell engineered to expressseveral specific genes are then implanted into the patient to allow forthe amelioration of the symptoms of diseases such as, but not limitedto, vascular, genitourinary tract, hernia or gastrointestinal disease.The gene expression may be under the control of a non-inducible (i.e.,constitutive) or inducible promoter. The level of gene expression andthe type of gene regulated can be controlled depending upon thetreatment modality being followed for an individual patient.

The use of the three-dimensional culture in gene therapy has a number ofadvantages. Firstly, since the culture comprises eukaryotic cells, thegene product will be properly expressed and processed in culture to forman active product. Secondly, gene therapy techniques are useful only ifthe number of transfected cells can be substantially enhanced to be ofclinical value, relevance, and utility; the three-dimensional culturesof the invention allow for expansion of the number of transfected cellsand amplification (via cell division) of transfected cells.

A variety of methods may be used to obtain the constitutive or transientexpression of gene products engineered into the stromal cells. Forexample, the transkaryotic implantation technique described by Seldon,R. F., et al., 1987, Science 236:714-718 can be used. "Transkaryotic",as used herein, suggests that the nuclei of the implanted cells havebeen altered by the addition of DNA sequences by stable or transienttransfection. The cells can be engineered using any of the variety ofvectors including, but not limited to, integrating viral vectors, e.g.,retrovirus vector or adeno-associated viral vectors, or non-integratingreplicating vectors, e.g., papilloma virus vectors, SV40 vectors,adenoviral vectors; or replication-defective viral vectors. Wheretransient expression is desired, non-integrating vectors and replicationdefective vectors may be preferred, since either inducible orconstitutive promoters can be used in these systems to controlexpression of the gene of interest. Alternatively, integrating vectorscan be used to obtain transient expression, provided the gene ofinterest is controlled by an inducible promoter.

Preferably, the expression control elements used should allow for theregulated expression of the gene so that the product is synthesized onlywhen needed in vivo. The promoter chosen would depend, in part upon thetype of tissue and cells cultured. Cells and tissues which are capableof secreting proteins (e.g., those characterized by abundant roughendoplasmic reticulum, and golgi complex) are preferable. Hosts cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.) and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow in an enriched media, and then are switched to aselective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which, in turn,can be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which express the geneprotein product.

Any promoter may be used to drive the expression of the inserted gene.For example, viral promoters include but are not limited to the CMVpromoter/enhancer, SV 40, papillomavirus, Epstein-Barr virus, elastingene promoter and β-globin. If transient expression is desired, suchconstitutive promoters are preferably used in a non-integrating and/orreplication-defective vector. Alternatively, inducible promoters couldbe used to drive the expression of the inserted gene when necessary. Forexample, inducible promoters include, but are not limited to,metallothionein and heat shock protein.

Examples of transcriptional control regions that exhibit tissuespecificity for connective tissues which have been described and couldbe used, include but are not limited to: elastin or elastase I genecontrol region which is active in pancreatic acinar cells (Swit et al.,1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp.Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515). Thedeposition of elastin is correlated with specific physiological anddevelopmental events in different tissues, including the vasculargrafts. For example, in developing arteries, elastin deposition appearsto be coordinated with changes in arterial pressure and mechanicalactivity. The transduction mechanisms that link mechanical activity toelastin expression involve cell-surface receptors. Onceelastin-synthesizing cells are attached to elastin through cell-surfacereceptors, the synthesis of additional elastin and other matrix proteinsmay be influenced by exposure to stress or mechanical forces in thetissue (for example, the constant movement of the construct in thebioreactor) or other factors that influence cellular shape.

Once genetically engineered cells are implanted into an individual, thepresence of TPA, streptokinase or urokinase activity can bring aboutamelioration of platelet aggregation, blood coagulation orthromboembolism. This activity is maintained for a limited time only,for example, to prevent potential complications that generally developduring the early phase after valve implantation, such as, plateletaggregation, blood clotting, coagulation or thromboembolism.Alternatively, once genetically engineered cells are implanted into anindividual, the presence of the anti-inflammatory gene products, forexample, peptides or polypeptides corresponding to the idiotype ofneutralizing antibodies for TNF, IL-2, or other inflammatory cytokines,can bring about amelioration of the inflammatory reactions associatedwith diseases such as vascular, gastrointestinal, hernia orgenitourinary tract disease.

The stromal cells used in the three-dimensional culture system of theinvention may be genetically engineered to "knock out" expression offactors or surface antigens that promote clotting or rejection at theimplant site. Negative modulatory techniques for the reduction of targetgene expression levels or target gene product activity levels arediscussed below. "Negative modulation", as used herein, refers to areduction in the level and/or activity of target gene product relativeto the level and/or activity of the target gene product in the absenceof the modulatory treatment. The expression of a gene native to stromalcell can be reduced or knocked out using a number of techniques, forexample, expression may be inhibited by inactivating the gene completely(commonly termed "knockout") using the homologous recombinationtechnique. Usually, an exon encoding an important region of the protein(or an exon 5' to that region) is interrupted by a positive selectablemarker (for example neo), preventing the production of normal mRNA fromthe target gene and resulting in inactivation of the gene. A gene mayalso be inactivated by creating a deletion in part of a gene, or bydeleting the entire gene. By using a construct with two regions ofhomology to the target gene that are far apart in the genome, thesequences intervening the two regions can be deleted. Mombaerts, P., etal., 1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084-3087.

Antisense and ribozyme molecules which inhibit expression of the targetgene can also be used in accordance with the invention to reduce thelevel of target gene activity. For example, antisense RNA moleculeswhich inhibit the expression of major histocompatibility gene complexes(HLA) shown to be most versatile with respect to immune responses. Stillfurther, triple helix molecules can be utilized in reducing the level oftarget gene activity. These techniques are described in detail by L. G.Davis, et al., eds, Basic Methods in Molecular Biology, 2nd ed.,Appleton & Lange, Norwalk, Conn. 1994.

In another alternative, the stromal cells can be genetically engineeredto block gene expression necessary for the transition of smooth musclecells to proliferate, migrate and to lead to development of neointimalhyperplasia, e.g., by antisense oligodeoxynucleotide blockade ofexpression of cell division cycle 2 kinase and proliferating cellnuclear antigen. Mann, M. J., et al., 1995, Proc. Natl. Acad. Sci.U.S.A. 92:4502-4506.

Using any of the foregoing techniques, the expression of fibrinogen, vonWillebrands factor, factor V or any cell surface molecule that binds tothe platelet α2Bβ-3 receptor can be knocked out in the stromal cells toreduce the risk of clot formation in the vascular or other types ofbiological tissue grafts. Likewise, the expression of MHC class IImolecules can be knocked out in order to reduce the risk of rejection ofthe graft.

In yet another embodiment of the invention, the three-dimensionalculture system could be used in vitro to produce biological products inhigh yield. For example, a cell which naturally produces largequantities of a particular biological product (e.g., a growth factor,regulatory factor, peptide hormone, antibody, etc.), or a host cellgenetically engineered to produce a foreign gene product, could beclonally expanded using the three-dimensional culture system in vitro.If the transformed cell excretes the gene product into the nutrientmedium, the product may be readily isolated from the spent orconditioned medium using standard separation techniques (e.g., HPLC,column chromatography, electrophoretic techniques, to name but a few). A"bioreactor" has been devised which takes advantage of the flow methodfor feeding the three-dimensional cultures in vitro. Essentially, asfresh media is passed through the three-dimensional culture, the geneproduct is washed out of the culture along with the cells released fromthe culture. The gene product is isolated (e.g., by HPLC columnchromatography, electrophoresis, etc.) from the outflow of spent orconditioned media.

The three-dimensional culture system of the invention may also afford avehicle for introducing genes and gene products in vivo for use in genetherapies or to augment healing at the site of implantation. Forexample, using recombinant DNA techniques, a gene for which a patient isdeficient could be placed under the control of a viral ortissue-specific promoter. Alternatively, DNA encoding a gene productthat enhances wound healing may be engineered into the cells grown inthe three-dimensional system. The recombinant DNA construct containingthe gene could be used to transform or transfect a host cell which iscloned and then clonally expanded in the three-dimensional culturesystem. The three-dimensional culture which expresses the active geneproduct, could be implanted into an individual who is deficient for thatproduct.

The use of the three-dimensional culture in gene therapy has a number ofadvantages. Firstly, since the culture comprises eukaryotic cells, thegene product will be properly expressed and processed in culture to forman active product. Secondly, gene therapy techniques are useful only ifthe number of transfected cells can be substantially enhanced to be ofclinical value, relevance, and utility; the three-dimensional culturesof the invention allow for expansion of the number of transfected cellsand amplification (via cell division) of transfected cells. For example,genetically engineered cells that express the gene product could beincorporated into living stromal tissue tubes that can be used as bloodvessels; in this case the gene product may be delivered to thebloodstream where it will circulate. Alternatively, geneticallyengineered cells that express wound healing factors may be incorporatedinto the living stromal cultures used to make tendons and ligaments toenhance wound healing at the site of implantation.

Preferably, the expression control elements used should allow for theregulated expression of the gene so that the product is synthesized onlywhen needed in vivo. The promoter chosen would depend, in part upon thetype of tissue and cells cultured. Cells and tissues which are capableof secreting proteins (e.g., those characterized by abundant roughendoplasmic reticulum and golgi complex) are preferable. To this end,liver and other glandular tissues could be selected. When using livercells, liver specific viral promoters, such as hepatitis B viruselements, could be used to introduce foreign genes into liver cells andregulate the expression of such genes. These cells could then becultured in the three-dimensional system of the invention.Alternatively, a liver-specific promoter such as the albumin promotercould be used.

Examples of transcriptional control regions that exhibit tissuespecificity which have been described and could be used, include but arenot limited to: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,1987, Hepatology 7:42S-51S); insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-122);immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al.,1984, Cell 38:647-658;.Adams et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444);albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276); alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58);alpha-1-antitrypsin gene control region which is active in liver (Kelseyet al., 1987, Genes and Devel. 1:161-171); beta-globin gene controlregion which is active in myeloid cells (Magram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94); myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Shani, 1985,Nature 314:283-286); and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

In a further embodiment of the invention, three-dimensional culturesmaybe used to facilitate gene transduction. For example, and not by wayof limitation, three-dimensional cultures of fibroblast stromacomprising a recombinant virus expression vector may be used to transferthe recombinant virus into cells brought into contact with the stromalmatrix, thereby simulating viral transmission in vivo. Thethree-dimensional culture system is a more efficient way ofaccomplishing gene transduction than are current techniques for DNAtransfection.

In an alternate embodiment of the invention, the three-dimensionalcultures may be used as model systems for the study of physiologic orpathologic conditions and the effect of drugs and treatments. Forexample, in a specific embodiment of the invention, a three-dimensionalculture system may be used as a model for the blood-brain barrier; sucha model system can be used to study the penetration of substancesthrough barriers such as the blood-brain barrier, the glomerularapparatus, and mucosa of nasopharyngeal passage lining.

For purposes of description only, and not by way of limitation, sampleembodiments of the invention are described below. For purposes ofdescription only, and not by way of limitation, the formation of thethree-dimensional cultures into tubes is described based upon the typeof tissue and cells used in various systems. These descriptionsspecifically include but are not limited to tubular sections ofgastrointestinal tract, genitourinary tract as well as blood vessels. Itis expressly understood that the three-dimensional culture system can beused with other types of cells to form other types of tubular tissues,all of which tissues are encompassed by the invention.

6. TUBULAR BIOLOGICAL TISSUES

The three-dimensional culture system can be used to construct single andmulti-layer tubular tissues in vitro. In accordance with the invention,these tubular structures can simulate tubular tissues and organs in thebody, including, but not limited to, blood vessels, gastrointestinaltract and genitourinary tract.

The different biological structures described below have severalfeatures in common. They are all tubular structures primarily composedof layers of stromal tissue with an interior lining of epithelium(gastrointestinal and genitourinary) or endothelium (blood vessels).Their connective tissues also contain layers of smooth muscle withvarying degrees of elastic fibers, both of which are especiallyprominent in arterial blood vessels. By including and sustaining thesecomponents in three-dimensional cultures according to the presentinvention, the tissues they compose can attain the special structuraland functional properties they require for proper physiologicalfunctioning in vivo. They can then serve as replacements for damaged ordiseased tubular tissues in a living body.

6.1. Single Mesh Layer Tubes

The following subsections describe the use of a mesh framework tosupport the growth of the living stromal tissue used to prepare tubesthat can be implanted into the body.

6.1.1. Flat Mesh Starting Material

A mesh can be cut into a rectangular strip of which the width isapproximately equal to the inner circumference of the tubular organ intowhich it will ultimately be inserted. The cells can be inoculated ontothis mesh and incubated by floating or suspending in liquid media. Atthe appropriate stage of confluence, the mesh can be rolled up into atube by joining the long edges together. The seam can be closed bysuturing the two edges together using fibers of a suitable material ofan appropriate diameter.

6.1.2. Tubular Mesh Starting Material

According to the invention, a mesh can be woven as a tube, inoculatedwith stromal cells and suspended in media in an incubation chamber. Inorder to prevent cells from occluding the lumen, one of the open ends ofthe tubular mesh can be affixed to a nozzle. Liquid media can be forcedthrough this nozzle from a source chamber connected to the incubationchamber to create a current through the interior of the tubular mesh.The other open end can be affixed to an outflow aperture which leadsinto a collection chamber, from which the media can be recirculatedthrough the source chamber. The tube can be detached from the nozzle andoutflow aperture when incubation is complete. This method is describedby Ballermann, B. J., et al., Int. Application No. WO 94/25584 and in apending application entitled, "APPARATUS AND METHOD FOR STERILIZING,SEEDING, CULTURING, STORING, SHIPPING AND TESTING TISSUE, SYNTHETIC, ORNATIVE VASCULAR GRAFTS," filed Apr. 27, 1995 by Peterson, A., et al.(Advanced Tissue Sciences, Inc.), Ser. No. 08/430,768, both of which areincorporated herein by reference in its entirety.

6.2. Multiple Mesh Layers Tubes

In general, two three-dimensional cultures can be combined into a tubein accordance with the invention using any of the following methods.

6.2.1. Multiple Flat Meshes

One flat rectangular culture can be laid atop another and suturedtogether. This two-layer sheet can then be rolled up, as described abovefor a single culture in Section 6.1.1, by joining together the longedges, and securing with sutures.

6.2.2. Flat Meshes Wrapped Around Tubular Meshes

One tubular mesh that is to serve as the inner layer can be inoculatedand incubated. A second mesh can be grown as a flat, rectangular stripwith width slightly larger than the outer circumference of the tubularmesh. After appropriate growth is attained, the rectangular mesh can bewrapped around the outside of the tubular mesh. Closing the seam of theouter strip and securing it to the inner tube can be accomplished in asingle suturing step.

6.2.3. Multiple Tubular Meshes

Two tubular meshes of slightly differing diameters can be grownseparately. The culture with the smaller diameter can be inserted insidethe larger one, and secured with sutures. This method would not bepractical for very narrow tubes.

For each of these methods, more layers can be added by reapplying themethod to the double layered tube. The meshes can be combined at anystage of growth of the culture they contain, and incubation of thecombined meshes can be continued when desirable.

According to the present invention, any suitable method can be employedto shape the three-dimensional culture to take on the conformation ofthe natural organ or tissue to be simulated.

The descriptions which follow are provided to demonstrate how toconstruct model tubular tissues and organs in vitro. In each case, oneor more stromal layers can be established as described in section 5.1.Particular attention is paid to generating the specialized properties ofspecific natural connective tissues by including and maintaining thematerials inherent to those natural tissues in the three-dimensionalcultures. One or more surface layers of generally more homogenouscellular composition (such as endothelium, epithelium, or smooth muscle)can then be cultured onto the stromal layer as described in Section 5.2.Using the methods outlined above in this section, thesethree-dimensional cultures can be shaped to assume a tubularconformation which simulates the shape of a natural tubular organ ortissue. Variations of this basic approach can be used to better simulatethe natural organs and tissues to be corrected.

These tubular constructions simulate biological structures in vivo andmay be readily implanted to replace damaged or diseased tissues.However, the invention encompasses the three-dimensional culturesdescribed herein in any possible form and does not require that thesecultures be formed into tubes. Flat three-dimensional cultures can beimplanted, for example, directly into the body to replace any part orall of the circumference of a tubular structure, depending on the extentof replacement required.

7. BLOOD VESSELS

The replication of blood vessel elements in vitro is described below inparticular for arteries and veins.

7.1. Arteries

Arteries are tubes lined with a thin layer of endothelial cells andgenerally composed of three layers of connective tissue: the intima(which is not present in many muscular arteries, particularly smallerones), media, and adventitia, in order from inside to outside.

The main cellular component of the inner two layers is anundifferentiated smooth muscle cell, which produces the extracellularprotein elastin. The internal elastic lamina, which lies just interiorto the media, is a homogenous layer of elastin. The abundance of elastinin their walls gives arteries the ability to stretch with everycontraction of the heart. The intima and media also contain somefibroblasts, monocytes, and macrophages, as well as some collagen.

The adventitia is composed of more ordinary connective tissue with bothelastic and collagenic fibers. Collagen in this layer is import ant inpreventing over-stretching.

While all the layers of the arterial wall are connective tissue, thereis a compositional and functional difference between the adventitia andthe inner two coats, the intima and the media. Consequently, it may beadvantageous in accordance with the invention to grow these differentlayers in separate meshes. Whether the intima and media are grown inseparate meshes, or combined in one, depends on how distinct theselayers are in the particular artery into which the three-dimensionalculture is to be implanted.

For example, according to the invention fibroblasts can be isolated fromthe adventitia of a patient's artery and used to inoculate athree-dimensional matrix, as described in Section 6.1, and grown tosubconfluence. Cells can be isolated from tissue rich inelastin-producing undifferentiated smooth muscle cells, also containingsome fibroblasts, from the intima and media of the same artery. Thesecells can be used to inoculate a separate mesh and grown tosubconfluence. Once the elastin-producing cells have proliferated to theappropriate extent, these meshes can be combined using one of themethods detailed in Section 6.2. In this manner, the smooth muscle cellscan proliferate and produce elastin in a three-dimensional environmentthat simulates that of natural arterial walls.

Endothelial cells can be isolated from the same patient. When the twocultures reach the appropriate degree of confluence, the endothelialcells can be seeded on top of the upper, elastin-rich layer andincubated until they form a confluent layer.

If a fully functional replacement with all the various layers of tissueis not required, a simple homogenous three-dimensional elastin-richstromal culture can be used. Alternatively, the stromal culture could belined with endothelium. More layers of this homogenous stromal matrixcan be combined to provide the appropriate thickness for such aprosthesis.

7.2. Veins

The layers of the connective tissue comprising the walls of veins areless well delineated than those of arteries, and contain much morecollagen and less elastin. Consequently, a single three-dimensionalculture can be grown, for example, from a single inoculum of cells.These cells consisting mostly of fibroblasts with some smooth musclecells, can be isolated from the walls of a vein of the patient. When theappropriate degree of confluence is reached, endothelial cells, isolatedfrom the same patient, for example, can be seeded on top of the stromallayer and grown to confluence.

8. GASTROINTESTINAL TRACT

Another embodiment of the invention provides for the replication ofgastrointestinal tract elements in vitro in a system comparable tophysiological conditions. The gastrointestinal tract comprises severaldifferent organs, but all have the same general histological scheme.

1. Mucous Membrane: The mucous membrane is the most interior layer ofthe gastrointestinal tract, and is composed of three sub-layers. Theabsorptive surfaces particularly are highly folded to increase thesurface area. The lumen is lined with a thin layer of epithelium, whichis surrounded by the lamina propria, a connective tissue which containsfibroblasts, some smooth muscle, capillaries, as well as collagenic,reticular, and some elastic fibers. Lymphocytes are also found here toprotect against invasion, especially at absorptive surfaces where theepithelium is thin. The third sub-layer, the muscularis mucosa, consistsof two thin layers of smooth muscle with varying amounts of elasticfibers. The smooth muscle fibers of the inner layer are arrangedcircularly, and the outer layer is arranged longitudinally.

2. Submucosa: This layer consists of loose connective tissue includingelastic fibers as well as larger blood vessels and nerve fibers.

3. Muscularis Externa: This layer consists of two thick layers of smoothmuscle which provide the motion which advances material along throughthe gastrointestinal tract. The muscle fibers of the inner layer arearranged circularly, while in the outer layer they are longitudinal. Anexception is the upper third of the esophagus, which contains striatedmuscle allowing for the voluntary contractions associated withswallowing.

4. Serosa (or Adventitia): This outermost layer consists of looseconnective tissue, covered by squamous mesothelium where the tract issuspended freely.

These four layers can be constructed in vitro in accordance with theinvention by making different three-dimensional tubular tissue cultures.For example, in order to construct the mucous membrane, a mesh composedof bioabsorbable material can be inoculated with fibroblasts, smoothmuscle cells, and other cells isolated from the lamina propria of thepatient who is to receive the implant, from a section of tract in oraround the site that is to be replaced.

If the site of transplantation is an absorptive surface, the mesh can becontoured on the surface which is to face the lumen.

Simultaneously, a second mesh whose inner circumference is slightlylarger than the outer circumference of the first mesh can be inoculatedwith the fibroblasts and other cells of the patient's submucosal layer.Similarly, a third mesh can be inoculated with cells from the serosa.These meshes can be configured and incubated as outlined in section 7.1.

When each stromal layer has grown to the appropriate extent, therespective surface layers can be cultured. For example, epithelial cellscan be seeded onto the top (or interior, if already tubular) of thelamina propria mesh, and smooth muscle cells can be seeded onto thebottom (or exterior, if already tubular) of the same mesh to form themuscularis mucosa.

In parallel, cells isolated from the muscularis externa can be seededonto the surface of the submucosa, or the surface of the serosa, orboth. Alternatively, the inner layer of the muscularis externa can begrown on the submucosal stromal mesh, and the outer layer can be grownon the serosal stromal mesh.

At appropriate stages of growth, these meshes can be combined using oneof the methods outlined in Section 7.2. The cultures can be incubateduntil the surface layers are mature.

During the assembly of the different three-dimensional cultures,vascular tissue (i.e., arteries and veins) can be added to the tubularconstruct. For example, a blood vessel can be constructed in vitro asoutlined in Section 8. Before combining the submucosal and serosalmeshes, this blood vessel can be laid down longitudinally along one orboth surfaces of the submucosal stromal culture. Upon implantation, itcan be spliced to the appropriate blood vessel of the adjoining segmentof the gastrointestinal tract.

By growing these layers separately, and then combining them and allowingfurther growth, distinct tissue layers can be formed and then allowed tomature in the same type of environment as naturally allows for theirspecialization.

In cases where only one of these layers has been damaged in the patient,a single three-dimensional culture would suffice, and can be implantedselectively to replace just that layer.

If a fully functional replacement with all the various layers of tissueis not required, a simple homogenous three-dimensional stromal culturelined with epithelium can be used. More layers of this homogenousstromal matrix can be combined to provide the appropriate thickness forsuch a prosthesis.

9. GENITOURINARY TRACT

Another embodiment of the invention provides for the replication ofgenitourinary tract elements in vitro in a system comparable tophysiological conditions. The genitourinary tract is very similar to thedigestive tract in terms of histology. The primary differences can bethe smaller diameters and lack of absorptive surface of thegenitourinary vessels.

9.1. Ureter

Like the gastrointestinal tract, the ureter also has a mucous membraneas its inner layer. Despite not having an absorptive surface, theinterior surface of the ureter is highly folded to form a stellateconformation in cross-section. The epithelial lining, however, is fourto five cells thick. The lamina propria, which lies beneath theepithelium, contains abundant collagen, some elastin, and occasionallymph nodules.

Surrounding the mucous membrane is a muscular coat, whose inner layercontains longitudinally arranged smooth muscle fibers, while those ofthe outer layer are circularly arranged. The outermost layer, theadventitia, consists of fibroelastic connective tissue.

In order to construct a simulation of a ureter in accordance with theinvention, stromal cells can be isolated from the two connective tissuesassociated with the ureter and used to initiate two separatethree-dimensional cultures as described in Section 6.1. Afterappropriate growth of the stromal layers, epithelial cells can be seededon the interior side of the lamina propria derived culture, and smoothmuscle cells can be seeded onto the opposite surface. In parallel,smooth muscle cells can be seeded onto one surface of the adventitiaderived culture. The two three-dimensional cultures can then be combinedto form one tubular structure, as described in Section 6.2., andincubated until the surface layers are mature.

If a fully functional replacement with all the various layers of stromaltissue is not required, a simple homogenous three-dimensional stromalculture lined with epithelium can be used. More layers of thishomogenous stromal matrix can be combined to provide the appropriatethickness for such a prosthesis.

9.2. Urethra

The urethra consists simply of a lamina propria which is lined withepithelium and surrounded by two layers of smooth muscle fibers. In theinner layer, the fibers are arranged longitudinally, while in the outerlayer they are circular. The connective tissue of the lamina propria isrich in elastic fibers and contains many venules.

Since the urethra has only one stromal layer, a single three-dimensionalculture may suffice for its construction in vitro in accordance with theinvention. A mesh can be inoculated, for example, with cellular materialisolated from the patient's urethral lamina propria as described insection 7.1. At the appropriate stage of confluence, epithelial cellscan be seeded onto one surface (interior) and smooth muscle can beseeded onto the opposite surface (exterior). The three-dimensionalculture can be incubated until the surface layers are mature.

10. HERNIA REPAIR

In herniorrhaphy, a corrective bioresorbable three-dimensional mesh,seeded with fibroblast cells could be used. Alternatively, cells mightbe seeded onto a synthetic mesh substrate for stronger fascial closure.

11. FORMATION OF TENDONS AND LIGAMENTS

Ligaments and tendons consist of fibroblasts surrounded by fibers ofcollagen type I and III and a predominance of the glycosaminoglycandermatan sulfate. The embodiment of the invention provides for theplacement of stromal tissue under mechanical or pulsatile forces toalter the formation and alignment of collagen fibers into bundles moredense and parallel than those routinely seen in dermis. By placingdermal fibroblasts on polymers and growing the tissues under increasingpulsing mechanical force, the final structure will have the tensilestrength of a normal tendon (≈33 MPa). Ligamentous or tendinousstructures are also created utilizing similar methods with the option ofattaching tissue-engineered bone to the end of the forming ligament ortendon in order to provide an attachment site.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

What is claimed is:
 1. A three-dimensional pancreatic culture comprisingpancreatic parenchymal cells cultured on a living stromal tissueprepared in vitro, comprising stromal cells and connective tissueproteins naturally secreted by the stromal cells attached to andsubstantially enveloping a framework composed of a biocompatible,non-living material formed into a three-dimensional structure havinginterstitial spaces bridged by the stromal cells.
 2. Thethree-dimensional cell culture of claim 1 in which the parenchymal cellsare pancreatic endocrine cells.
 3. The three-dimensional cell culture ofclaim 2 in which the pancreatic endocrine cells are islet of Langerhanscells.
 4. The three-dimensional cell culture of claim 1 in which theparenchymal cells are pancreatic exocrine cells.
 5. Thethree-dimensional pancreatic culture of claim 1 in which the pancreaticparenchymal cells comprise a combination of exocrine and endocrinecells.
 6. The three-dimensional cell culture of claim 5 in which thepancreatic exocrine cells are acinar cells.
 7. The three-dimensionalcell culture of claims 1, 2, 3, 4, or 6 in which the parenchymal cellsare genetically engineered cells.
 8. The three-dimensional cell cultureof claim 1 in which the stromal cells are fibroblasts.
 9. Thethree-dimensional cell culture of claim 1 in which the stromal cells area combination of fibroblasts and endothelial cells, pericytes,macrophages, monocytes, leukocytes, plasma cells, mast cells oradipocytes.
 10. The three-dimensional cell culture of claim 1 in whichthe framework is composed of a biodegradable material.
 11. Thethree-dimensional cell culture of claim 10 in which the biodegradablematerial is cotton, polyglycolic acid, cat gut sutures, cellulose,gelatin, or dextran.
 12. The three-dimensional cell culture of claim 1in which the framework is composed of a non-biodegradable material. 13.The three-dimensional cell culture of claim 12 in which thenon-biodegradable material is a polyamide, a polyester, a polystyrene, apolypropylene, a polyacrylate, a polyvinyl, a polycarbonate, apolytetrafluorethylene, or a nitrocellulose compound.
 14. Thethree-dimensional cell culture of claim 10, 11, 12 or 13 in which theframework is pre-coated with collagen.
 15. The three-dimensional cellculture of claim 1, 2, 3, 4, 6, 8, 9, 10 or 13 in which the framework isa mesh.
 16. The three-dimensional cell culture of claim 7 in which theframework is a mesh.
 17. The three-dimensional cell culture of claim 14in which the framework is a mesh.
 18. The three-dimensional pancreaticculture of claim 1 in which the pancreatic parenchymal cells comprisepancreatic stem cells.
 19. The three-dimensional pancreatic culture ofclaim 1 in which the stromal cells comprise umbilical cord cells,placental cells, mesenchymal stem cells or fetal cells.
 20. A method forculturing pancreatic cells in vitro, comprising culturing pancreaticparenchymal cells inoculated onto a living stromal tissue prepared invitro, comprising the stromal cells and connective tissue proteinsnaturally secreted by the stromal cells attached to and substantiallyenveloping a framework composed of a biocompatible, non-living materialformed into a three-dimensional structure having interstitial spacesbridged by the stromal cells.
 21. The method of claim 20 in which theparenchymal cells are pancreatic endocrine cells.
 22. The method ofclaim 21 in which the pancreatic endocrine cells are islet of Langerhanscells.
 23. The method of claim 20 in which the parenchymal cells arepancreatic exocrine cells.
 24. The method of claim 20 in which thepancreatic parenchymal cells comprise a combination of exocrine andendocrine cells.
 25. The method of claim 24 in which the pancreaticexocrine cells are acinar cells.
 26. The method of claim 20, 21, 22, 23or 25 in which the parenchymal cells are genetically engineered cells.27. The method according to claim 20 in which the stromal cells arefibroblasts.
 28. The method according to claim 20 in which the stromalcells are a combination of fibroblasts and endothelial cells, pericytes,macrophages, monocytes, leukocytes, plasma cells, mast cells oradipocytes.
 29. The method according to claim 20 in which the frameworkis composed of a biodegradable material.
 30. The method according toclaim 29 in which the biodegradable material is cotton, polyglycolicacid, cat gut sutures, cellulose, gelatin, or dextran.
 31. The methodaccording to claim 20 in which the framework is composed of anon-biodegradable material.
 32. The method according to claim 31 inwhich the non-biodegradable material is a polyamide, a polyester, apolystyrene, a polypropylene, a polyacrylate, a polyvinyl, apolycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.33. The method according to claim 29, 30, 31, or 32 in which theframework is pre-coated with collagen.
 34. The method according to claim20, 21, 22, 23, 25, 27, 28, 29, 30, 31 or 32 in which the framework is amesh.
 35. The method according to claim 26 in which the framework is amesh.
 36. The method according to claim 33 in which the framework is amesh.
 37. The method of claim 20 in which the pancreatic parenchymalcells comprise pancreatic stem cells.
 38. The method of claim 20 inwhich the stromal cells comprise umbilical cord cells, placental cells,mesenchymal stem cells or fetal cells.